The lack of effective treatment for a number of neurological diseases, many of which are fatal, is a significant public health concern. Approximately four million Americans have Alzheimer's disease (AD) and the number will increase to approximately fourteen million by the year 2005, unless a cure or prevention is found. Similarly, amyotrophic lateral sclerosis (ALS), commonly known as Lou Gherig's disease, is a fatal neurodegenerative disease that affects approximately 2 per 100,000 people. In the U.S. alone, more than 5,000 people are diagnosed with ALS each year. Approximately 15 million people in the U.S. suffer from diabetes, and most are at risk for development of neuropathy associated with diabetes. The incidence of diabetic neuropathy is considered to be 6 in 10,000 people. Down syndrome (DS), which occurs in 1 of 1,000 live births, also causes severe developmental brain abnormalities. Moreover, DS patients develop AD by their fifth decade of life. The common denominator for neurodegenerative disorders (which also include Parkinson's disease (PD) and Huntington disease (HD)) is selective neuronal loss due to programmed cell death or apoptosis. To date, there are no known cures or effective treatments for these diseases. Acute neurological diseases involving apoptotic death offer a window of opportunity for short-term treatment. They include stroke, trauma and hypoxia-ischemia, where there is a delayed “secondary” brain injury in a “penumbra zone”, that is initially spared and surrounds the area of most severe damage. Cell death in these penumbra areas is due to apoptosis and is amenable to anti-apoptotic therapeutic strategies that minimize brain damage. While stroke is the third common cause of death in the US, it is a leading cause of long-term disability. Over 400,000 subjects suffer from a first ischemic stroke each year in the US. Effective treatment is not currently available, and gene therapy is limited by the paucity of genes with anti-apoptotic activity in the CNS.
Following spinal cord injury (SCI), loss of motor neurons occurs by mechanical tissue disruption as well as necrosis. Secondarily, degeneration results from a cascade of events triggered by the injury and results in the activation of endogenous apoptosis, cell death. Apoptosis does not occur immediately after injury; rather it occurs over a prolonged period of several weeks. The cause of the apoptosis includes loss of trophic support, intracellular oxidative stress including oxidative damage to, and activation of caspases including caspase-q and caspase-3 in both neurons and microglia. Moreover an increase in the expression of pro-apoptotic peptide Bax and a reciprocal decrease in the anti-apoptotic Bcl-2 peptide in the mitochondrial-enriched membrane compartments occurs with SCI. Such a loss of motor neurons causes paralysis and death.
Apoptosis is a normal physiological process observed in many cell types and enables useful pruning of “mismatched” or excessive cells during development and maturation. Apoptosis is critical to modeling of the nervous system during embryonic development and in the regulation and function of the immune system. However, cellular homeostasis is dependent on a proper balance between survival/proliferation and apoptotic processes. Thus, excessive apoptosis can lead to non-physiological death, and ultimately to disease states. In the adult nervous system, where there is little cell production and little cell death, excessive apoptosis results in neurological diseases including AD, ALS, DS, PD, and HD, or is subsequent to physical ischemic or chemical injury of the central nervous system (CNS). What exactly prompts apoptosis in chronic disorders is unclear, but several stimuli are thought to play an etiologic role. Such stimuli include oxidative stress that may increase with age, loss of neurotrophic support, accumulated burden of endogenous and exogenous factors, and excessive release of neurotransmitters known as excitotoxins.
Features associated with apoptosis include cell shrinkage, exposure of phosphatidylserine on the outer surface of the membrane, plasma membrane blebbing, mitochondrial dysfunction, DNA cleavage, chromatin condensation, and formation of membrane bound apoptotic bodies. Apoptosis involves three phases: (i) a time-variable phase called commitment which refers to the time from the reception of apoptotic stimuli to the irreversible initiation of the second phase, (ii) execution phase in which all of the dramatic changes associated with cell death occur, and (iii) clearance, which involves engulfment of apoptotic bodies by neighboring cells or be professional phagocytes without the stimulation of an inflammatory response. The commitment phase is influenced by the proliferative status of the cell, cell type, apoptotic stimulus and expression of regulatory genes that inhibit or promote apoptosis. During the commitment phase, a cell is faced with multiple decision/check points. The nature and intensity of the incoming stimulus may determine whether the cell survives (if the defense mechanisms can overcome the insult) or commits to undergo a series of apoptotic phases. This death is characterized by several morphological changes including condensation of the cell nucleus and membrane blebbing. In addition, there is DNA fragmentation caused by a group of caspases (cysteine aspartases) that are specifically activated in apoptotic cells by proteolytic cleavage. Indeed, the execution process of apoptosis occurs mainly via activation of a series of proteolytic enzymes, termed caspases. Once active, caspases cleave a number of cellular proteins including proteins involved in DNA repair and replication such as poly (ADP-ribose) polymerase (PARP), DNA-dependent protein kinase (DNA-PK), and Inhibitors of Caspase-Activated DNAse (ICAD), as well as structural proteins, thereby inducing endonuclease activation and the characteristic morphological changes associated with apoptosis.
Caspase-3 is one of the key executioners of apoptosis. Its activation requires proteolytic cleavage of the inactive procaspase-3 into activated 17-20 kDa and 12 kDa subunits. Activated caspase-3 is, in turn, responsible, either partially or totally, for the proteolytic cleavage of many key proteins, such as PARP, that is involved in DNA repair. PARP cleavage is a crucial event in the commitment to undergo apoptosis. Again, cell homeostasis depends on the balance between apoptotic and survival/proliferation processes. Survival stimuli cause the membrane bound G protein, Ras, to adopt an active, GTP-bound state, and it, in turn, coordinates the activation of a multitude of downstream effectors. The mitogenic/survival Ras/MEK/MAPK pathway begins with the activation of Raf kinase and is followed by the activation of MAP kinase (MEK) and mitogen activated protein kinase (MAPK). A variety of genes, including those required for cell cycle progression, are targets for MAPK. The Ras/MEK/MAPK pathway is also involved in the control of apoptosis, presumably by upregulating anti-apoptotic proteins such as bcl-2 or mcl-1 (Bonni, et. al., 1999, Science, 286:1358-1362).
Herpes Simplex Virus Type 2 (HSV-2) is a dual tropic virus that infects cells of the mucosal epithelium as well as cells of the nervous system. HSV-2 encodes a ribonucleotide reductase (RR) enzyme comprised of two subunits, referred to as the large and small subunits, encoded by the UL39 and UL40 genes, respectively. Within the large submit of HSV-2 RR (ICP10), resides a protein kinase domain termed ICP10PK whose sequence is known in the art. ICP10PK exhibits intrinsic protein kinase activity and has previously been shown to cause neoplastic transformation in a variety of cell types.
Viruses depend on cells for their replication and they can differenitially affect various signaling pathways. Herpes Simplex Virus Type 1 (HSV-1) and HSV-2 can trigger or counteract apoptosis in a cell-specific manner (Aubert et al., 1999, J. Virol., 73:2803-2813; Aubert et al., 1999, J. Virol., 73:10359-10370; Chou et al., 1992, Proc. Natl. Acad. Sci. USA, 89:3266-3270). Anti-apoptotic activity was ascribed to the HSV-1 and HSV-2 gene US3, and to the HSV-1 genes γ134.5, US5, ICP27, and LAT (Aubert et al., 1999, J. Virol., 73:2803-2813; Chou et al., 1992, Proc. Natl. Acad. Sci. USA, 89:3266-3270). However, their exact mechanism of action and their activity in hippocampal neurons, if any, are still poorly understood.
There is a long felt need for treatment programs which arrest or alleviate neurological diseases where the diseases are caused, at least in part, by apoptosis of neuronal cells. The present invention satisfies this need.